Antibodies and Proteases as Probes of the Structures of Mammalian Nucleoside Transporters

Kwong, F. Y. P.; Fincham, H. E.; Davies, Anthony; Henderson, Peter J. F.; Baldwin, S. A.; Young, James D.

doi:10.1007/978-1-4899-2638-8_98

Cited by 10 publications

(9 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…mENT1, like hENT1, has a single putative glycosylation site in the first extracellular loop (Asn-48). This coincides with the finding that photoaffinity-labelled mENT1 transporters migrate on SDS\polyacrylamide gels with an apparent M r of 45000 [39], which is close to the size of the hENT1 transporter (M r 45000-55000) but much smaller than the rENT1 transporter (M r " 62000), which has three potential glycosylation sites [40]. This glycosylated residue has been implicated in the binding of NBMPR to hENT1, as mutation of this residue to glutamine resulted in a 10-fold decrease in the affinity of NBMPR for the mutant transporter expressed in yeast [41].…”

Section: Ment1supporting

confidence: 87%

Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain

Kiss

Farah

Kim

et al. 2000

Biochemical Journal

View full text Add to dashboard Cite

Mammalian cells express at least two subtypes of equilibrative nucleoside transporters, i.e. ENT1 and ENT2, which can be distinguished functionally by their sensitivity and resistance respectively to inhibition by nitrobenzylthioinosine. The ENT1 transporters exhibit distinctive species differences in their sensitivities to inhibition by dipyridamole, dilazep and draflazine (human mouse rat). A comparison of the ENT1 structures in the three species would facilitate the identification of the regions involved in the actions of these cardioprotective agents. We now report the molecular cloning and functional expression of the murine (m)ENT1 and mENT2 transporters. mENT1 and mENT2 encode proteins containing 458 and 456 residues respectively, with a predicted 11-transmembrane-domain topology. mENT1 has 88 % and 78 % amino acid identity with rat ENT1 and human ENT1 respectively ; mENT2 is more highly conserved, with 94 % and 88 % identity with rat ENT2 and human ENT2 respectively. We have also isolated two additional distinct cDNAs that encode proteins similar to mENT1 ; these probably represent distinct mENT1 isoforms or alternative splicing products. One cDNA encodes a protein with two additional amino acids

show abstract

Section: Ment1supporting

confidence: 87%

Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain

Kiss

Farah

Kim

et al. 2000

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…The inability of [ 3 H]NBMPR to photolabel mENT1⌬11 suggests that the loss of this C-terminal region of mENT1 removes the residue that NBMPR normally cross-links to or that a critical residue has shifted (Kwong et al, 1993), we would argue that the loss of the last three transmembrane domains of mENT1 leads to a conformation change that prevents covalent attachment of the [ 3 H]N-BMPR to elements of its binding pocket in the N-terminal part of the protein. It is generally believed that the S-nitrobenzyl group of NBMPR is photoactivated upon exposure to UV light; hence the amino acid residue involved in the photoaffinity labeling is probably proximal to the mENT1 region that binds the S-nitrobenzyl moiety of NBMPR (Paterson and Oliver, 1971;Young et al, 1983;Shi et al, 1984;Zhu et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Characterization of mENT1Δ11, a Novel Alternative Splice Variant of the Mouse Equilibrative Nucleoside Transporter 1

Robillard

Bone²,

Park³

et al. 2008

Mol Pharmacol

View full text Add to dashboard Cite

Mammalian cells require specific transport mechanisms for the cellular uptake and release of endogenous nucleosides such as adenosine, and nucleoside analogs used in chemotherapy. We have identified a novel splice variant of the mouse equilibrative nucleoside transporter, mENT1, that results from the exclusion of exon 11 during pre-RNA processing. This variant encodes a truncated protein (mENT1⌬11) missing the last three transmembrane domains of the full-length mENT1. The mENT1⌬11 transcript and protein were found to be differentially distributed among tissues relative to full-length mENT1. PK15-NTD (nucleoside transport deficient) cells were transfected with mENT1 or mENT1⌬11 and assessed for nucleoside transport function. No significant differences were observed between the mENT1 and mENT1⌬11 in terms of transport function or inhibitor binding affinity. PK15-mENT1⌬11 transfected cells bound the ENT1 3 H]uridine. The only significant differences between the mENT1 variants were that mENT1⌬11 could not be photolabeled with [3 H]NBMPR and that mENT1⌬11 was insensitive to the transporter-modifying effects of N-ethylmaleimide. These data suggest that the last three transmembrane domains of mENT1 are not necessary for transport activity, but this region does contain the cysteines responsible for the sensitivity of mENT1 to sulfhydryl reagents, and the residues important for covalent modification of the protein with NBMPR. These results provide important guidelines for future mutagenesis studies aimed at elucidating the tertiary structure of the ENT1 protein and the domains involved in inhibitor binding and substrate translocation.

show abstract

“…Earlier studies had established that the es transporter of human erythrocytes, which is readily identified by site-specific photolabeling of plasma membranes with 3 H-NBMPR (Jarvis and Young, 1987), is glycosylated and migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the "band 4.5" region with the erythrocyte glucose transporter (Wu et al, 1983;Kwong et al, 1992). Because the glycosylation site(s) of the erythrocyte es transporter are known to be near one of its ends (Kwong et al, 1993), current topology models are based on the assumption that Asn 48 is the site of N-linked glycosylation, thereby placing Loop 1 at the extracellular membrane face (Griffiths et al, 1997a). Glycosylation of Loop 1 and its consequent external orientation has recently been confirmed in studies in which the glycosylation site (Asn 48) of hENT1 was eliminated by site-directed mutagenesis (Sundaram, M., Yao, S. Y. M., Chomey, E., Baldwin, S. A., Cass, C. E., and Young, J. D., unpublished results).…”

Section: The Entl (Es) Subfamilymentioning

confidence: 99%

Nucleoside Transporters of Mammalian Cells

Cass

Young

Baldwin

et al. 2002

Pharmaceutical Biotechnology

121

View full text Add to dashboard Cite

Antibodies and Proteases as Probes of the Structures of Mammalian Nucleoside Transporters

Cited by 10 publications

References 7 publications

Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain

Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain

Characterization of mENT1Δ11, a Novel Alternative Splice Variant of the Mouse Equilibrative Nucleoside Transporter 1

Nucleoside Transporters of Mammalian Cells

Contact Info

Product

Resources

About